WO2024003102A1 - Méthodes pour prévenir, retarder la progression ou traiter la cholestase et/ou la fibrose associée à la cholestase - Google Patents

Méthodes pour prévenir, retarder la progression ou traiter la cholestase et/ou la fibrose associée à la cholestase Download PDF

Info

Publication number
WO2024003102A1
WO2024003102A1 PCT/EP2023/067574 EP2023067574W WO2024003102A1 WO 2024003102 A1 WO2024003102 A1 WO 2024003102A1 EP 2023067574 W EP2023067574 W EP 2023067574W WO 2024003102 A1 WO2024003102 A1 WO 2024003102A1
Authority
WO
WIPO (PCT)
Prior art keywords
sirna
claudin
agent
antibody
seq
Prior art date
Application number
PCT/EP2023/067574
Other languages
English (en)
Inventor
Felix BAIER
Deborah KEOGH-STROKA
Daniel CANDINAS VON ALBERTINI
Nicolas MELIN
Original Assignee
Universität Bern
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universität Bern filed Critical Universität Bern
Publication of WO2024003102A1 publication Critical patent/WO2024003102A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • the present invention relates to a method of prevention, delay of progression or treatment of cholestasis and/or fibrosis associated with cholestasis.
  • Cholestasis is characterized by a reduction or stagnation of bile flow in the liver. This can be caused either by obstruction of extrahepatic bile ducts, or by intrahepatic defects in bile synthesis or circulation.
  • the aetiology includes drug intoxications, alcoholic or viral hepatitis, biliary atresia, gallstones and genetic diseases.
  • Cholestatic liver diseases are a major personal and economic burden for patients and society; liver dysfunction due to cholestasis accounts for about 10 % of all liver transplantations that are performed in Europe 1.
  • liver dysfunction due to cholestasis accounts for about 10 % of all liver transplantations that are performed in Europe 1.
  • responsible for this intriguingly high number is the distressing absence of effective treatment options for cholestatic diseases like primary sclerosing cholangitis (PSC) or primary biliary cirrhosis (PBC).
  • PSC primary sclerosing cholangitis
  • PBC primary biliary cirrhosis
  • the current first line of treatment involves the administration of the immunomodulatory- and bicarbonate secretion stimulating drug ursodeoxycholic acid (UDCA), which improves transplantation free survival in about 60% of PBC patients and shows only limited efficacy on PSC patients 2.
  • UDCA immunomodulatory- and bicarbonate secretion stimulating drug ursodeoxycholic acid
  • New therapeutic approaches are focussed on immunomodulatory strategies 3–5, microbiota alterations 6,7 or activation of FGF19/FXR signalling for anti-fibrotic effects and improvements of bile acid export and detoxification 8–10.
  • PPAR agonists for the treatment of cholestasis 11–13 to increase the efflux of toxic bile acids.
  • the treatment of chronic cholestatic diseases is difficult and often ineffective.
  • New treatments regarding cholestasis and/or fibrosis associated with cholestasischolestasis and/or fibrosis associated with cholestasis are necessary in order to meet the high medical need.
  • the present invention provides an agent which inhibits the expression and/or activity of Claudin-3 for use in a method of prevention, delay of progression or treatment of cholestasis and/or fibrosis associated with cholestasis.
  • the present invention provides a composition comprising an agent which inhibits the expression and/or activity of Claudin-3 and a pharmaceutically acceptable carrier for use in a method of prevention, delay of progression or treatment of cholestasis and/or fibrosis associated with cholestasis.
  • the present invention provides a dosage form for the prevention, delay of progression or treatment of cholestasis and/or fibrosis associated with cholestasis, comprising an agent which inhibits the expression and/or activity of Claudin-3 or a composition comprising said agent, and a pharmaceutically acceptable carrier.
  • the present invention provides a siRNA targeting Claudin-3. Brief description of the figures Figure 1.
  • Claudin-3 knockout leads to impaired bile acid metabolism, diluted bile and impaired uptake of the fluorescent tracer FITC Dextran.
  • A Metascape analysis.
  • D Serum ALT levels.
  • E Serum AST levels.
  • Figure 3. Reduced bile acid concentration in liver and gallbladder bile following common bile duct ligation (BDL)
  • B TBA quantification in bile following BDL.
  • C TBA quantification in liver bile extracts following BDL.
  • D Serum markers of cholestasis and liver injury after BDL and anti claudin-3 GalNAc siRNA injection.
  • the present invention relates to a method of prevention, delay of progression or treatment of cholestasis and/or fibrosis associated with cholestasis.
  • the present invention provides an agent which inhibits the expression and/or activity of Claudin-3 for use in a method of prevention, delay of progression or treatment of cholestasis and/or fibrosis associated with cholestasis.
  • the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa.
  • the blockage can occur in the liver (intrahepatic cholestasis) or in the bile ducts (extrahepatic cholestasis).
  • cholestasis denotes an impairment of bile flow and failure to secrete the inorganic and organic constituents of bile.
  • cholestasis arises from molecular and ultrastructural changes that impair the entry of small organic molecules, inorganic salts, proteins and ultimately water into the biliary space.
  • the physical findings of jaundiceand pruritus are accompanied by elevated serum concentrations of bilirubin, bile salts and alkaline phosphatase (ALP).
  • Cholestasis can be caused by extrahepatic issues, such as a gallstone or tumour blocking the flow of bile outside of the liver. But it can also have intrahepatic causes like viral diseases, genetic disorders and bile duct strictures.
  • Bile constitutes the primary pathway for elimination of bilirubin, excess cholesterol (both as free cholesterol and as bile salts) and xenobiotics that are insufficiently water soluble to be excreted into urine.
  • a fundamental driver of bile formation is hepatocellular secretion of bile salts into the canalicular space, which entrains secretion of phosphatidylcholine and cholesterol from the hepatocyte.
  • Bile facilitates the digestion and absorption of lipids from the gut. Because bile formation requires well-functioning hepatocytes and an intact biliary tree, this process is readily disrupted.
  • fibrosis associated with cholestasis refers to any fibrotic liver disease that is associated to cholestasis and/or that is initially caused by liver cholestasis.
  • agent which inhibits the expression and/or activity of Claudin-3 refers to any biological or chemical agent which permits inhibition of the expression and/or inhibition of the activity of Claudin-3 e.g. by reducing or disrupting interactions of Claudin-3 or its gene with other biomolecules, such as but not limited to protein-protein interaction, ligand-receptor interaction, or protein- nucleic acid interaction.
  • agents include, but are not limited to, antibodies, protein-binding agents, nucleic acid molecules, small molecules, recombinant proteins, peptides, aptamers, avimers and protein-binding derivatives, or fragments thereof.
  • Claudin-3 can be inhibited, e.g., by antibodies binding Claudin-3 e.g. antibodies binding at least one of the extracellular domains of Claudin-3 or toxins which bind to Claudin-3.
  • Expression of Claudin-3 can be inhibited, e.g., by a DNA targeting agent (e.g., CRISPR system, TALE, Zinc finger protein) or an RNA targeting agent (e.g., inhibitory nucleic acid molecules).
  • Inhibition of expression of Claudin-3 comprises a decrease of expression of at least 10%, preferably of at least 40% in the presence of the agent compared to the expression of Claudin-3 without the agent.
  • Inhibition of activity of Claudin-3 comprises a decrease of acitivity of at least 10%, preferably of at least 40% in the presence of the agent compared to the activity of Claudin-3 without the agent.
  • Claudins as referred herein are a family of integral membrane proteins that make up TJs, which are the chief intercellular junctions that act as permeability barriers and confer polarity to epithelial cells by demarcating the membrane upper and lower regions.
  • the mammalian claudin family comprises 27 proteins, and many alternative splicing claudin proteins are expressed in various tissues. In the past decade, the crystal structures of this protein family have been gradually elucidated.
  • Claudins are tetratransmembrane proteins, including four transmembrane domains (TM1-4), the intracellular N and C termini, and two extracellular loops (ECL1 and ECL2).
  • ECL1 contains four ⁇ -strands and an extracellular helix (ECH)
  • ECL2 contains a ⁇ -strand and cell surface-exposed transmembrane 3 domain.
  • the ECLs are involved in the formation of interactions between claudin strands and determine the gate function of claudin-based TJs by two variable regions.
  • Claudin-3 was originally termed rat ventral prostate 1 protein (RVP1), and Clostridium perfringens enterotoxin receptor 2 (CPETR2).
  • Claudin-3 refers to the mammalian, preferably to the human Claudin-3 with the Uniprot (www.uniprot.org) identifier Uniprot.
  • toxin which binds to Claudin-3 refers to a peptide that binds to Claudin-3, like CPE (Clostridium perfringens enterotoxin) or a fragment or variant thereof, such as C-terminal fragments of Clostridium perfringens enterotoxin (cCPE), for example as shown in doi: 10.1074/jbc.M111.312165.
  • CPE Clostridium perfringens enterotoxin
  • fragment or variant thereof in relation to the CPE is meant that the fragment or variant (such as a cCPE analogue, derivative or mutant) is capable of binding to a extracellular domain of claudin-3, in order to inhibit claudin-3 binding to another protein.
  • Immunoglobulins are generally comprising four polypeptide chains, two heavy (H) chains and two light (L) chains, and are therefore multimeric proteins, or an equivalent Ig homologue thereof (e.g., a camelid nanobody, which comprises only a heavy chain, single domain antibodies (dAbs) which can be either be derived from a heavy or light chain); including full length functional mutants, variants, or derivatives thereof (including, but not limited to, murine, chimeric, humanized and fully human antibodies, which retain the essential epitope binding features of an Ig molecule, and including dual specific, bispecific, multispecific, and dual variable domain immunoglobulins; Immunoglobulin molecules can be of any class (e.g., IgG, IgE, IgM, IgD, IgA
  • the antibody used in the present invention is preferably a monoclonal antibody or a fragment thereof and comprises modified antibody formats and antibody mimetics, in particular a human or humanized antibody.
  • An "antibody fragment”, as used herein, relates to a molecule comprising at least one polypeptide chain derived from an antibody that is not full length, including, but not limited to (i) a Fab fragment, which is a monovalent fragment consisting of the variable light (VL), variable heavy (VH), constant light (CL) and constant heavy 1 (CHI) domains; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a heavy chain portion of a Fab(Fa) fragment, which consists of the VHand CHI domains; (iv) a variable fragment (Fv) fragment, which consists of the VLand VHdomains of a single arm of an antibody, (v) a domain
  • modified antibody format encompasses antibody-drug- conjugates, Polyalkylene oxide-modified scFv, Monobodies, Diabodies, Camelid Antibodies, Domain Antibodies, bi- or trispecific antibodies, IgA, or two IgG structures joined by a J chain and a secretory component, shark antibodies, new world primate framework + non-new world primate CDR, IgG4 antibodies with hinge region removed, IgG with two additional binding sites engineered into the CH3 domains, antibodies with altered Fc region to enhance affinity for Fc gamma receptors, dimerised constructs comprising CH3+VL+VH, and the like.
  • antibody mimetic refers to proteins not belonging to the immunoglobulin family, and even non-proteins such as aptamers, or synthetic polymers. Some types have an antibody-like beta-sheet structure. Potential advantages of "antibody mimetics” or “alternative scaffolds” over antibodies are better solubility, higher tissue penetration, higher stability towards heat and enzymes, and comparatively low production costs.
  • conjugation or “conjugated”, as used herein, relates to the covalent or non-covalent binding of a molecule to another molecule. Covalent binding includes formation of a covalent bond. Non-covalent binding includes p-p (aromatic) interactions, van der Waals interactions, H-bonding interactions, and ionic interactions.
  • a conjugate comprising covalent binding of the present invention is e.g. a N-acetylgalactosamine (GalNAc) siRNA conjugate wherein siRNA, e.g. siRNA targeting Claudin-3 is covalently bound to N-acetylgalactosamine (GalNAc), preferably covalently bound to one- to five moieties of N-acetylgalactosamine (GalNAc), more preferably covalently bound to three moieties of N-acetylgalactosamine (GalNAc).
  • a conjugate comprising non-covalent binding of the present invention is e.g.
  • nucleic acid is a polymer comprising or consisting of nucleotide monomers, which are covalently linked to each other by phosphodiester-bonds of a sugar/phosphate-backbone.
  • nucleic acid sequence also encompasses modified nucleic acid sequences, such as base-modified, sugar-modified or backbone-modified etc., DNA or RNA.
  • nucleic acid targeting a gene or mRNA refers to at least one nucleic acid sequence encoding or comprising a nucleic acid like small interfering RNA (siRNA), short or small harpin RNA (shRNA), microRNA (miRNA), piwi-interacting RNA (piRNA), and long non-coding RNA (IncRNA).
  • siRNA small interfering RNA
  • shRNA short or small harpin RNA
  • miRNA microRNA
  • piRNA piwi-interacting RNA
  • IncRNA long non-coding RNA
  • a small interfering RNA (siRNA) is capable of binding to a target gene or a target messenger RNA (mRNA).
  • siRNAs as used herein may be processed from a dsRNA or a shRNA.
  • siRNA as used herein, may encompass a siRNA according to the invention and a molecule, in particular a dsRNA molecule, from which a siRNA according to the invention can be generated within a mammalian cell by the RNA interference pathway.
  • the RNA may be made by synthetic chemical and enzymatic methodology known to one of ordinary skill in the art, or by the use of recombinant technology, or may be isolated from natural sources, or by a combination thereof.
  • RNA may optionally comprise unnatural and naturally occurring nucleoside modifications known in the art such as e.g., N1-Methylpseudouridine also referred as methylpseudouridine.
  • the nucleic acid targeting a gene or mRNA of the present invention comprises multiple copies of siRNAs that can target one mRNA.
  • siRNA binding Claudin-3 relates to a small interfering RNA (siRNA) capable of binding to a target messenger RNA (mRNA) of Claudin-3.
  • siRNAs as used herein may comprise a double-stranded RNA (dsRNA) region, a hairpin structure, a loop structure, or any combinations thereof.
  • siRNAs may comprise at least one shRNA, at least one dsRNA region, or at least one loop structure.
  • siRNAs may be processed from a dsRNA or an shRNA.
  • siRNAs may be processed or cleaved by an endogenous protein, such as DICER, from an shRNA.
  • DICER an endogenous protein
  • a hairpin structure or a loop structure may be cleaved or removed from an siRNA.
  • a hairpin structure or a loop structure of an shRNA may be cleaved or removed.
  • RNAs described herein may be made by synthetic, chemical, or enzymatic methodology known to one of ordinary skill in the art, made by recombinant technology known to one of ordinary skill in the art, or isolated from natural sources, or made by any combinations thereof.
  • the RNA may comprise modified or unmodified nucleotides or mixtures thereof, e.g., the RNA may optionally comprise chemical and naturally occurring nucleoside modifications known in the art.
  • siRNA may comprise a nucleic acid sequence comprising a sense siRNA strand.
  • siRNA may comprise a nucleic acid sequence comprising an anti-sense siRNA strand.
  • siRNA may comprise a nucleic acid sequence comprising a sense siRNA strand and a nucleic acid sequence comprising an anti-sense siRNA strand.
  • the term "compound which factilitates delivery of the agent to the liver” or “compound which factilitates delivery of the siRNA to the liver” as used herein, relates to e.g. a sugar, a lipid nanoparticle, a liposome, or a adenovirus.
  • a compound which factilitates delivery of the siRNA to the liver is e.g. N-acetylgalactosamine (GalNAc), which is preferred.
  • GalNAc N-acetylgalactosamine
  • the subject is a mammal. Mammals include, but are not limited to primates (including human and non-human primates). In a preferred embodiment, the subject is a human.
  • pharmaceutically acceptable carrier refers to carriers that are suitable for use with humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio.
  • Carriers can be solvents, suspending agents or vehicles, for delivering the instant agents to a subject.
  • the term "about” as used herein refers to +/- 10% of a given measurement.
  • the present invention provides an agent which inhibits the expression and/or activity of Claudin-3 for use in a method of prevention, delay of progression or treatment of cholestasis and/or fibrosis associated with cholestasis.
  • Agent which inhibits the expression and/or activity of Claudin-3 In one embodiment the agent which inhibits the expression and/or activity of Claudin-3 is a siRNA targeting Claudin-3 or an antibody or a fragment thereof which binds to Claudin-3. In one embodiment the agent which inhibits the expression and/or activity of Claudin-3 is an agent which inhibits the expression of Claudin-3.
  • the agent which inhibits the expression of Claudin-3 is a nucleic acid targeting a gene or mRNA coding for Claudin-3.
  • Nucleic acids targeting a gene coding for Claudin-3 or targeting a mRNA coding for Claudin-3 can be e.g. at least one nucleic acid sequence encoding or comprising a nucleic acid like small interfering RNA (siRNA), short or small harpin RNA (shRNA), microRNA (miRNA), piwi- interacting RNA (piRNA), and long non-coding RNA (IncRNA).
  • the agent which inhibits the expression of Claudin-3 is a small interfering RNA (siRNA) targeting Claudin-3 i.e.
  • siRNA targeting Claudin-3 comprises 10-50, more preferably 15- 40, even more preferably 17-24 nucleotides.
  • siRNA according to the invention comprises 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 nucleotides.
  • the siRNA targeting Claudin-3 comprises the sequence as shown in SEQ ID NO:1 (sense strand), siRNA comprising the sequence as shown in SEQ ID NO: 2 (sense strand), siRNA which is at least 95% identical, more preferably 96%, 97%, 98%, 99% or 100% identical to the siRNA comprising the sequence as shown in SEQ ID NO:1 and siRNA which is at least 95% identical, more preferably 96%, 97%, 98%, 99% or 100% identical to the siRNA comprising the sequence as shown in SEQ ID NO: 2.
  • the siRNA targeting Claudin-3 is characterized by a sequence reverse complementary to SEQ ID NO:1 or by a sequence reverse complementary to SEQ ID NO:2.
  • the sequence reverse complementary to SEQ ID NO:1 is the sequence as shown in SEQ ID NO: 5 (anti-sense strand) and the sequence reverse complementary to SEQ ID NO:2 is the sequence as shown in SEQ ID NO: 6 (anti-sense strand).
  • the siRNA targeting Claudin-3 is selected from the group consisting of siRNA comprising the sequence as shown in any of SEQ ID NOs: 1, 2, 5, 6 or 33-168 or siRNA which is at least 95% identical, more preferably 96%, 97%, 98%, 99% or 100% identical to the siRNA comprising the sequence as shown in any of SEQ ID NO: 1, 2, 5, 6 or 33-168.
  • the siRNA targeting Claudin-3 is selected from the group consisting of siRNA comprising the sequence as shown in any of SEQ ID NOs: 33-168 or siRNA which is at least 95% identical, more preferably 96%, 97%, 98%, 99% or 100% identical to the siRNA comprising the sequence as shown in any of SEQ ID NO: 33-168.
  • the siRNA targeting Claudin-3 selected from the group consisting of siRNA comprising the sequence as shown in any of SEQ ID NOs: 33- 100 or siRNA which is at least 95% identical, more preferably 96%, 97%, 98%, 99% or 100% identical to the siRNA comprising the sequence as shown in any of SEQ ID NO: 33-100.
  • siRNA targeting Claudin-3 is selected from the group consisting of siRNA comprising the sequence as shown in any of SEQ ID NOs: 1, 2, 33-66 or 101-134 siRNA which is at least 95% identical, more preferably 96%, 97%, 98%, 99% or 100% identical to the siRNA comprising the sequence as shown in any of SEQ ID NO: 1, 2, 33-66 or 101-134.
  • siRNA targeting Claudin-3 is selected from the group consisting of siRNA comprising the sequence as shown in any of SEQ ID NOs: 33-66 or 101-134 or siRNA which is at least 95% identical, more preferably 96%, 97%, 98%, 99% or 100% identical to the siRNA comprising the sequence as shown in any of SEQ ID NO: 33-66 or 101-134.
  • siRNA targeting Claudin-3 selected from the group consisting of siRNA comprising the sequence as shown in any of SEQ ID NOs: 33- 66 or siRNA which is at least 95% identical, more preferably 96%, 97%, 98%, 99% or 100% identical to the siRNA comprising the sequence as shown in any of SEQ ID NO: 33-66.
  • sequence identity and percentage of sequence identity refer to the values determined by comparing two aligned sequences. Methods for alignment of sequences for comparison are well-known in the art. Alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl.
  • the agent which inhibits the expression and/or activity of Claudin-3 is an agent which inhibits the activity of Claudin-3.
  • the agent which inhibits the activity of Claudin-3 is selected from the group consisting of an antibody or a fragment thereof which binds to Claudin-3 and a toxin which binds to Claudin-3.
  • the toxin which binds to Claudin-3 is Clostridium perfringens enterotoxin (CPE) or a fragment or variant thereof.
  • the agent which inhibits the activity of Claudin-3 is an antibody or a fragment thereof which binds to Claudin-3, preferably a monoclonal antibody or a fragment thereof which binds to Claudin-3, even more preferably an antibody or a fragment thereof which binds to an extracellular domain of Claudin-3, in particular a monoclonal antibody or a fragment thereof which binds to an extracellular domain of Claudin-3.
  • the antibody or a fragment thereof which binds to Claudin-3 is a human or humanized antibody, more preferably a monoclonal human antibody.
  • the antibody or a fragment thereof which binds to Claudin-3 comprises a light chain comprising the amino acid sequence as shown in SEQ ID NO: 169 and/or a heavy chain comprising the amino acid sequence as shown in SEQ ID NO: 170.
  • the agent which inhibits the activity of Claudin-3 is an antibody or a fragment thereof, preferably a monoclonal antibody or a fragment thereof which binds to the extracellular loop 1 and/or 2 (ECL1 and/or ECL2) of claudin-3.
  • the agent which inhibits the expression and/or activity of Claudin-3 is conjugated to a compound which facilitates delivery of the agent to the liver.
  • a compound which facilitates delivery of the agent to the liver can be e.g. N-acetylgalactosamine (GalNAc), a lipid nanoparticle, a liposome or an adenovirus.
  • the antibody or a fragment thereof which binds to Claudin-3 is conjugated to a compound which facilitates delivery of the antibody to the liver, even more preferably, the antibody or a fragment thereof which binds to Claudin-3 is a N- acetylgalactosamine (GalNAc) antibody conjugate.
  • the siRNA targeting Claudin-3 is conjugated to a compound which facilitates delivery of the siRNA to the liver.
  • the agent is a N- acetylgalactosamine (GalNAc) siRNA conjugate i.e. a conjugate of a siRNA targeting Claudin- 3 and N-acetylgalactosamine (GalNAc).
  • a conjugate comprises 1-5, preferably 3 moiteties of GalNAc covalently bound to one moitety of siRNA.
  • compositions As outlined above, the invention also relates to a composition comprising an agent which inhibits the expression and/or activity of Claudin-3 and optionally a pharmaceutically acceptable carrier for use in a method of prevention, delay of progression or treatment of cholestasis and/or fibrosis associated with cholestasis.
  • composition refers usually to a fixed-dose combination (FDC) that includes the compound which inhibits the expression and/or activity of Claudin-3 in a single dosage form, having a predetermined combination of respective dosages.
  • the composition further may be used as add-on therapy.
  • additive-on or “add-on therapy” means an assemblage of reagents for use in therapy, the subject receiving the therapy begins a first treatment regimen of one or more reagents prior to beginning a second treatment regimen of one or more different reagents in addition to the first treatment regimen, so that not all of the reagents used in the therapy are started at the same time.
  • the amount of the agent which inhibits the expression and/or activity of Claudin-3 to be administered will vary depending upon factors such as the particular agent, disease condition and its severity, according to the particular circumstances surrounding the case, including, e.g., the specific agent which inhibits the expression and/or activity of Claudin-3 being administered, the route of administration, the condition being treated, the target area being treated, and the subject or host being treated.
  • the invention provides a composition comprising an agent which inhibits the expression and/or activity of Claudin-3, wherein said agent which inhibits the expression and/or activity of Claudin-3 is present in a therapeutically effective amount.
  • an amount capable of invoking one or more of the following effects in a subject receiving the composition of the present invention refers to an amount capable of invoking one or more of the following effects in a subject receiving the composition of the present invention: (i) dilution and/or- detoxification of the bile, which contains a higher amount of water than before the start of the therapy (ii) decrease of the concentration of liver-and/or blood bile acids (iii) decreased levels of the cholestasis marker alkaline phosphatase (ALP) in the blood (iv) less tissue necrosis in the liver (v) improved quality of life (vi) longer symptom free survival (vii) longer liver transplant free survival (viii) ameliorated cholestasis symptoms like jaundice or pruritus (ix) increased survival rate of patients with cholestasis and/or fibrosis associated with cholestasis.
  • ALP alkaline phosphatase
  • the invention provides a composition comprising agent which inhibits the expression and/or activity of Claudin-3, wherein the amount of said agent which inhibits the expression and/or activity of Claudin-3 in the composition is from about 0.1 mg to about 10g.
  • Formulations and modes of administration A composition according to the invention is, preferably, suitable for subcutaneous, intra- venous or oral administration to a subject and comprises a therapeutically effective amount of the active ingredient and one or more suitable pharmaceutically acceptable carrier.
  • compositions like pharmaceutical compositions, which are preferred can be formulated in a conventional manner using one or more pharmaceutically acceptable inactive ingredients, like pharmaceutical carriers that facilitate processing of the active compounds into preparations that can be used pharmaceutically.
  • a proper formulation is dependent upon the route of administration chosen and a summary of pharmaceutical compositions can be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed.
  • a composition according to the invention is prepared in a manner known per se, e.g. by means of conventional mixing, granulating, coating, dissolving or lyophilizing processes.
  • any of the usual pharmaceutical media may be employed, for example water, glycols, oils, alcohols, carriers, such as starches, sugars, or microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like.
  • the invention provides a composition comprising an agent which inhibits the expression and/or activity of Claudin-3 and at least one pharmaceutically acceptable carrier, wherein the composition is a solution.
  • the invention provides a composition comprising an agent which inhibits the expression and/or activity of Claudin-3 and at least one pharmaceutically acceptable carrier, wherein the composition is, wherein the composition is a tablet or a capsule, preferably a tablet.
  • Dosing regimen An exemplary treatment regime entails administration once daily, twice daily, three times daily, every second day, twice per week, once per week.
  • the composition of the invention is usually administered on multiple occasions. Intervals between single dosages can be, for example, less than a day, daily, every second day, twice per week, or weekly.
  • the composition of the invention may be given as a continous uninterrupted treatment.
  • composition of the invention may also be given in a regime in which the subject receives cycles of treatment interrupted by a drug holiday or period of non-treatment.
  • the composition of the invention may be administered according to the selected intervals above for a continuous period of one week or a part thereof, for two weeks, for three weeks, for four weeks, for five weeks or for six weeks and then stopped for a period of one week, or a part thereof, for two weeks, for three weeks, for four weeks, for five weeks, or for six weeks.
  • the composition of the treatment interval and the non-treatment interval is called a cycle.
  • the cycle may be repeated one or more times. Two or more different cycles may be used in combination for repeating the treatment one or more times.
  • Intervals can also be irregular as indicated by measuring blood levels of said agent which inhibits the expression and/or activity of Claudin-3 in the patient.
  • the composition according to the invention is administered once daily.
  • the agent which inhibits the expression and/or activity of Claudin-3 can be administered from 0.1 mg – 10 g per day.
  • an agent which inhibits the expression and/or activity of Claudin-3 or a composition thereof to prevent, delay of progression or treat cholestasis and/or fibrosis associated with cholestasis
  • the present invention provides an agent which inhibits the expression and/or activity of Claudin-3 or a composition thereof as described herein, for use in a method of prevention, delay of progression or treatment cholestasis and/or fibrosis associated with cholestasis in a subject.
  • an agent which inhibits the expression and/or activity of Claudin-3 or a composition thereof as described herein for the manufacture of a medicament for the prevention, delay of progression or treatment of cholestasis and/or fibrosis associated with cholestasis in a subject. Also provided is the use of an agent which inhibits the expression and/or activity of Claudin-3 or a composition thereof as described herein for the prevention, delay of progression or treatment of cholestasis and/or fibrosis associated with cholestasis in a subject.
  • treatment includes: (1) delaying the appearance of clinical symptoms of the state, disorder or condition developing in an animal, particularly a mammal and especially a human, that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; (2) inhibiting the state, disorder or condition (e.g.
  • delay of progression means increasing the time from symptoms to worsening of symptoms of cholestasis and/or fibrosis associated with cholestasis and includes reversing or inhibition of disease progression.
  • inhibittion of disease progression or disease complication in a subject means preventing or reducing the disease progression and/or disease complication in the subject.
  • Preventive treatments comprise prophylactic treatments.
  • the composition of the invention is administered to a subject suspected of having, or at risk for developing cholestasis and/or fibrosis associated with cholestasis.
  • the composition is administered to a subject such as a patient already suffering from cholestasis and/or fibrosis associated with cholestasis s, in an amount sufficient to cure or at least partially arrest the symptoms of the disease. Amounts effective for this use will depend on the severity and course of the disease, previous therapy, the subject's health status and response to the drugs, and the judgment of the treating physician.
  • the composition of the invention may be administered chronically, which is, for an extended period of time, including throughout the duration of the subject's life in order to ameliorate or otherwise control or limit the symptoms of the subject's disease or condition.
  • the composition may be administered continuously; alternatively, the dose of drugs being administered may be temporarily reduced or temporarily suspended for a certain length of time (i.e., a “drug holiday”).
  • a maintenance dose of the composition of the invention is administered if necessary.
  • the dosage or the frequency of administration, or both is optionally reduced, as a function of the symptoms, to a level at which the improved disease is retained.
  • cholestasis is intrahepatic cholestasis or extrahepatic cholestasis, preferably intrahepatic cholestasis.
  • cholestasis is intrahepatic cholestasis wherein the intrahepatic cholestasis is acute intrahepatic cholestasis or chronic intrahepatic cholestasis.
  • cholestasis is chronic intrahepatic cholestasis, preferably chronic intrahepatic cholestasis selected from the group consisting of primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC).
  • PBC primary biliary cholangitis
  • PSC primary sclerosing cholangitis
  • cholestasis is a disease selected from the group consisting of obstructive cholestasis, nonobstructive cholestasis, bile duct diseases and defects in biliary function, more preferably bile duct diseases, in particular malignant bile duct obstructions.
  • cholestasis and/or fibrosis associated with cholestasis are selected from the group consisting of chronic intrahepatic cholestasis and bile duct diseases.
  • cholestasis and/or fibrosis associated with cholestasis are selected from the group consisting of primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC) and malignant bile duct obstructions.
  • PBC primary biliary cholangitis
  • PSC primary sclerosing cholangitis
  • malignant bile duct obstructions PBC
  • Obstructive cholestasis can be associated with or can be caused by gallstones, pancreatic cancer, bile duct adenocarcinoma, stricture of the common bile duct, biliary atresia like extrahepatic biliary atresia, common bile duct obstruction caused by like biliary sludge or gallstones, choledochal cyst with biliary sludge and/or inspissated bile/mucous plug.
  • Nonobstructive cholestasis can be associated with or can be caused by viral hepatitis (hepatitis B hepatitis C), toxic effects of drugs or nutrition, paraneoplastic syndrome like Hodgkin lymphoma, Wilson disease, familial cholestatic syndromes, intrahepatic cholestasis of pregnancy, infiltrative disorders like amyloidosis, ormetastatic cancer, and/or cirrhosis (any cause), bacterial infection like infections with gram-negative enteric bacteraemia, syphilis, listeria, toxoplasma, or sepsis or endotoxaemia caused by bacterial infection, viral infections with cytomegalovirus, herpesvirus (includes simplex, zoster, parvovirus B19, adenovirus), rubella, reovirus, and enteroviruses.
  • viral hepatitis hepatitis B hepatitis C
  • paraneoplastic syndrome like Hodgkin
  • Bile duct diseases can be associated with or can be caused by primary biliary cholangitis, primary sclerosing cholangitis, Graft-versus-host disease (acute and chronic), transplant rejection (acute and chronic), Transplant: infarction of the biliary tree secondary to hepatic artery obstruction, vanishing bile duct syndromes caused by toxic effects of drugs such as e.g. ibuprofen or chlorpromazine.
  • Defects in biliary function can be associated with or can be caused by alpha-1-antitrypsin storage disease, cystic fibrosis, galactosaemia, tyrosinaemia, fatty acid oxidation defects, lipid storage disorders, glycogen storage disorders, peroxisomal disorders, bile acid biosynthetic defects, PFIC1, PFIC2, PFIC3 (PFIC, Progressive familial intrahepatic cholestasis), pucity of bile ducts like Alagille syndrome or nonsyndromic paucity of bile duct syndromes.
  • alpha-1-antitrypsin storage disease cystic fibrosis, galactosaemia, tyrosinaemia, fatty acid oxidation defects, lipid storage disorders, glycogen storage disorders, peroxisomal disorders, bile acid biosynthetic defects, PFIC1, PFIC2, PFIC3 (PFIC, Progressive familial intrahepatic cholestasis), pucity of
  • cholestasis is a disease selected from the group consisting of obstructive cholestasis associated with or caused by stricture of the common bile duct, biliary atresia like extrahepatic biliary atresia and/or common bile duct obstruction caused by like biliary sludge or gallstones, preferably obstructive cholestasis associated with or caused by stricture of the common bile duct; nonobstructive cholestasis associated with or caused by toxic effects of drugs, Wilson disease and/or familial cholestatic syndromes; bile duct diseases associated with or caused by primary biliary cholangitis, primary sclerosing cholangitis and/or vanishing bile duct syndromes caused by toxic effects of drugs such as e.g.
  • cholestasis is obstructive cholestasis.
  • cholestasis is obstructive cholestasis associated with or caused by stricture of the common bile duct or biliary atresia like extrahepatic biliary atresia.
  • fibrosis associated with cholestasis is a disease where the fibrosis is at least in part induced by an unresolved cholestasis, preferably fibrosis associated with cholestasis is a liver disease where the fibrosis is at least in part induced by a unresolved cholestasis.
  • Fibrosis associated with cholestasis is usually selected from the diseases or conditions selected from the group consisting of fibrosis due to hepatitis A Virus infection associated with cholestasis, fibrosis due to hepatitis B Virus infection associated with cholestasis, fibrosis due to hepatitis C Virus infection associated with cholestasis, fibrosis due to Epstein-Barr-Virus infection associated with cholestasis, fibrosis due to hemochromatosis associated with cholestasis, fibrosis due to autoimmune hepatitis associated with cholestasis, fibrosis due to secondary biliary cirrhosis associated with cholestasis, fibrotic due to alcoholic liver disease associated with cholestasis and / or clinical jaundice, fibrosis due to drug induced liver disease associated with cholestasis, fibrosis caused by metabolic syndromes, e.g., impaired glucose metabolism
  • the present invention provides a treatment for fibrosis causing metabolic diseases like NAFLD or NASH, in particular a treatment for fibrosis causing liver diseases using the agent as described herein.
  • the present invention provides a dosage form for the prevention, delay of progression or treatment cholestasis and/or fibrosis associated with cholestasis, comprising an agent which inhibits the expression or activity of Claudin-3 or a composition comprising said agent, and optionally a pharmaceutically acceptable carrier.
  • the present invention provides a siRNA targeting Claudin-3.
  • the present invention provides siRNAs targeting Claudin-3 selected from the group consisting of siRNA comprising the sequence as shown in SEQ ID NO:1, siRNA comprising the sequence as shown in SEQ ID NO: 2, siRNA which is at least 95% identical, more preferably 96%, 97%, 98%, 99% or 100% identical to the siRNA comprising the sequence as shown in SEQ ID NO:1 and siRNA which is at least 95% identical, more preferably 96%, 97%, 98%, 99% or 100% identical to the siRNA comprising the sequence as shown in SEQ ID NO: 2.
  • the present invention provides siRNAs targeting Claudin-3 selected from the group consisting of siRNA comprising the sequence as shown in SEQ ID NO:5, siRNA comprising the sequence as shown in SEQ ID NO: 6, siRNA which is at least 95% identical, more preferably 96%, 97%, 98%, 99% or 100% identical to the siRNA comprising the sequence as shown in SEQ ID NO:5 and siRNA which is at least 95% identical, more preferably 96%, 97%, 98%, 99% or 100% identical to the siRNA comprising the sequence as shown in SEQ ID NO: 6.
  • the present invention provides siRNAs targeting Claudin-3 selected from the group consisting of siRNA comprising the sequence as shown in any of SEQ ID NOs: 1, 2, 5, 6 or 33-168 or siRNA which is at least 95% identical, more preferably 96%, 97%, 98%, 99% or 100% identical to the siRNA comprising the sequence as shown in any of SEQ ID NO: 1, 2, 5, 6 or 33-168.
  • the present invention provides siRNAs targeting Claudin-3 selected from the group consisting of siRNA comprising the sequence as shown in any of SEQ ID NOs: 33-168 or siRNA which is at least 95% identical, more preferably 96%, 97%, 98%, 99% or 100% identical to the siRNA comprising the sequence as shown in any of SEQ ID NO: 33-168.
  • the present invention provides siRNAs targeting Claudin-3 selected from the group consisting of siRNA comprising the sequence as shown in any of SEQ ID NOs: 33-100 or siRNA which is at least 95% identical, more preferably 96%, 97%, 98%, 99% or 100% identical to the siRNA comprising the sequence as shown in any of SEQ ID NO: 33-100.
  • the present invention provides siRNAs targeting Claudin-3 is selected from the group consisting of siRNA comprising the sequence as shown in any of SEQ ID NOs: 1, 2, 33-66 or 101-134 or siRNA which is at least 95% identical, more preferably 96%, 97%, 98%, 99% or 100% identical to the siRNA comprising the sequence as shown in any of SEQ ID NO: 1, 2, 33-66 or 101-134.
  • the present invention provides siRNAs targeting Claudin-3 is selected from the group consisting of siRNA comprising the sequence as shown in any of SEQ ID NOs: 33-66 or 101-134 or siRNA which is at least 95% identical, more preferably 96%, 97%, 98%, 99% or 100% identical to the siRNA comprising the sequence as shown in any of SEQ ID NO: 33-66 or 101-134.
  • the present invention provides siRNAs targeting Claudin-3selected from the group consisting of siRNA comprising the sequence as shown in any of SEQ ID NOs: 33-66 or siRNA which is at least 95% identical, more preferably 96%, 97%, 98%, 99% or 100% identical to the siRNA comprising the sequence as shown in any of SEQ ID NO: 33-66.
  • the present invention provides an antibody or a fragment thereof which binds to Claudin-3.
  • the antibody or a fragment thereof which binds to Claudin-3 is a human antibody.
  • the antibody or a fragment thereof which binds to Claudin-3 comprises a light chain comprising the amino acid sequence as shown in SEQ ID NO: 169 and/or a heavy chain comprising the amino acid sequence as shown in SEQ ID NO: 170.
  • the antibody or a fragment thereof which binds to Claudin-3 is conjugated to a compound which facilitates delivery of the antibody to the liver.
  • the antibody or a fragment thereof which binds to Claudin-3 is a N-acetylgalactosamine (GalNAc) antibody conjugate.
  • a particular preferred antibody is a human antibody which binds to Claudin-3, more particular a human antibody which binds to Claudin-3 which comprises a light chain comprising the amino acid sequence as shown in SEQ ID NO: 169 and/or a heavy chain comprising the amino acid sequence as shown in SEQ ID NO: 170 and which is a N-acetylgalactosamine (GalNAc) antibody conjugate.
  • a human antibody which binds to Claudin-3 which comprises a light chain comprising the amino acid sequence as shown in SEQ ID NO: 169 and/or a heavy chain comprising the amino acid sequence as shown in SEQ ID NO: 170 and which is a N-acetylgalactosamine (GalNAc) antibody conjugate.
  • GalNAc N-acetylgalactosamine
  • Bile flow, and FITC Dextran passage to bile The integrity of the paracellular barrier of hepatocyte tight junction was measured as previously described14. Quantification of the blood- to bile passage of FITC dextran (40 kDa) was measured. In anaestetized mice, the common bile duct was ligated, and the gallbladder was canulated. Bile was subsequently collected for 20 minutes to determine the bile flow rate. Then, 400 ⁇ l of FITC dextran (40 kDa, 25 mg/ml in saline) were injected into the inferior vena cava. Bile was collected from the canulation for 40 minutes, in 3-minute fractions. The liver was surgically removed and weighted.
  • Bile was diluted 1/50 in water, and FITC fluorescence was measured at excitation 489nm / emission 534nm, using a Tecan Spark® plate reader. Data is reported as relative fluorescent units (RFU) per ⁇ l bile per minute per g liver.
  • REU relative fluorescent units
  • Quantification of the uptake of FITC dextran In anaestetized mice, the common bile duct was ligated and 300 ⁇ l of FITC dextran (40 kDa, 6.25 mg/ml in saline) were injected via the tail vein. FITC fluorescence was acquired in a timeseries imaging using the IVIS® Spectrum system (Perkin Elmer). One image was acquired each 30 seconds, for a total of 45 minutes.
  • Regions of interest with same area size were drawn around the liver.
  • the FITC signal intensity was quantified in each image using the device software. Data is reported as total radient efficiency [p/s]/[ ⁇ W/cm2].
  • mouse livers were harvested at 20 minutes post FITC dextran (40 kDa) injection and cryosections (25 ⁇ m) were made. Sections were stained with DAPI (D9542, diluted 1:2000; Sigma-Aldrich) for 15 minutes and mounted withfluorescence mounting medium (H-1000; Vectorlabs, Burlin-game, CA).
  • the common bile duct was exposed and double ligated with 7-0 silk (SofsilkTM, Covidien / Medtronic, Dublin, Ireland) and cut in between the ligatures.
  • the laparotomy was closed with 6-0 Prolene® suture (Ethicon, Bridgewater, New Jersey, United states).
  • Buprenorphine analgesia was repeated if indicated based on animal health scoring.
  • Hematoxylin & Eosin staining Liver paraffin sections were deparaffinized stained with Haematoxylin (Merck, Cat. #HX43078349) for six minutes, and differentiated in HCL-ALC (1:1) performing three dips. Slides were incubated in Eosin (Fluka Chemical Corp, Cat.
  • the Sirius red signal was separated from the other channels using the deconvolution setting “Azon-Mallory”.
  • the threshold was set. After conversion of the threshold adjusted image into an 8-bit image, the signal intensity was measured as integrated density. Results are represented as average integrated density per mm2.
  • Masson trichrome staining and quantification Masson trichrome staining. Paraffi n-embedded liver tissue was dewaxed and placed in Bouin’sfi xative (HT10-1-32; Sigma-Aldrich) at 56°C for 10 minutes. After washing slides in tap water and distilled H 2 O, slides were stained with hematoxylin (HT10-79; Sigma-Aldrich) for 5 minutes.
  • slides were destained once with HCl-alcohol (1:1) and rinsed again in distilled H 2 O.
  • slides were put in Biebrich scarlet-scid fuchsin (HT151-250ML; Sigma-Aldrich) diluted 1:2 in 1% acetic acid (K45741563 425; Dr. Grogg Chemie, Stettlen, Switzerland) for 1 minute.
  • Slides were rinsed and stained with phosphomolybdic-phosphotungstic acid (HT153-250ML and HT152-250ML; Sigma) 1:1 for 5 minutes. Slides then were stained with Aniline Blue (HT154-250ML; Sigma) for 20 minutes.
  • ALP and total bilirubin likewise were measured on the Cobas 8000, using the module C702 (Roche, Switzerland). All measurements were performed following the manufacturer’s instructions. Quantification of kidney bile plugs 7 days post BDL kidneys were sectioned entirely, and tissue was stained with haematoxylin/eosin. Images were taken using a bright-field microscope (Panoramic 250 Flash III, 3DHISTECH). ImageJ software was used to deconvolute the yellow-colored bile plugs from the pink haematxilin/eosin staining. The yellow color was transformed to a black and white 8-bit image, and the signal intensity was measured as integrated density.
  • Flow Cytometry Antibodies used for fluorescence-activated cell sorting are listed in below. Livers were put in Roswell Park Memorial Institute (RPMI) 1640 Medium, supplemented with GlutaMAX and digested with collagenase D (Merck), 0.05% collagenase IV (Worthington Biochemical) and DNase I (Sigma-Aldrich) and Dispase for 25min at 37°C. The digested liver-tissue was poured through a 100 ⁇ m cell strainer (BD Falcon) and washed in 50ml RPMI at 4°C. After centrifugation at 300g for 5 min, the supernatant was discarded and refilled with 30ml RPMI.
  • RPMI Roswell Park Memorial Institute
  • Red Blood Cell (RBC) Lysis Buffer was added per sample and filtered through 40 ⁇ m cell strainer and incubated at room temperature. After incubation, the cell suspension was spun down and washed in 5ml PBS (Gibco) containing 3% fetal bovine serum and 2mM EDTA and Hepes (staining buffer). The isolated non-parenchymal cells were then incubated with purified anti- anti-CD16/CD32 and viability dye eFluor 506 (Thermo Fisher Scientific) diluted in PBS for 20 minutes at 4C in the dark to block non-specific binding antibodies and exclude dead cells.
  • PBS Red Blood Cell
  • eFluor 506 Thermo Fisher Scientific
  • the samples were stained with primary antibodies (Table 1) diluted in the staining buffer for 20 minutes at 4C in the dark. After the final washing step 300g for 5min, the samples were resuspended in the staining buffer and fixed by adding IC Fixations buffer (eBioscience). The fixed samples were assessed using the flow cytometer (BD LSR Fortessa; BD Pharmingen, Inc, San Diego, CA) using the corresponding BD FACS Diva software. Data analysis was performed using FlowJo software (Treestar, Inc, Ashland, OR). FACS antibodies Fluorescence Cell Marker Clone Company Catalog no.
  • Bile, serum, and total liver tissue samples were processed according to the manufacturer instructions. Final absorbance levels were measured on a Tecan Tecan Spark® plate reader. Bile acid quantification using LC-MS/MS The method applied was described recently17. Briefly, for quantification of bile acids, 25 ⁇ L serum samples diluted 1:4 with water, and calibrators were subjected to protein precipitation by adding 900 ⁇ L of 2-propanol and a mixture of deuterated internal standards. Extraction was performed for 30 min at 4 °C with continuous shaking, followed by centrifuging 16000 g for 10 min. Supernatants were transferred to new tubes, evaporated to dryness and reconstituted with 100 ⁇ L methanol:water (1:1, v/v).
  • liver samples For the extraction of liver samples, 900 ⁇ L of chloroform:methanol:water (1:3:1, v/v/v) and 100 ⁇ L internal standard mixture were added to a Precellys tube containing beads and 30 ⁇ 5 mg of liver tissue. Samples were homogenized with a Precellys tissue homogenizer, centrifuging 16000 g for 10 min at 20°C. The supernatant was transferred to a new tube and the procedure repeated by adding 800 ⁇ L of extraction solvent. After evaporation to dryness, samples were resuspended with 200 ⁇ L methanol:water (1:1, v/v). The injection volume was in both cases 3 ⁇ L.
  • LC-MS/MS consisted of an Agilent 1290 UPLC coupled to an Agilent 6490 triple quadrupole mass spectrometer equipped with an electrospray ionization source (Agilent Technologies, Basel, Switzerland). Chromatographic separation of bile acids was achieved using a reversed-phase column (ACQUITY UPLC BEH C18, 1.7 mm, 2.1 ⁇ m, 150 mm, Waters, Wexford, Ireland)18.
  • ANIT intoxication ⁇ -Naphtylisothiocyanate (ANIT) intoxication protocols are derived from previous publications19,20. In the acute intoxication model, ANIT (Sigma-Aldrich, St.
  • Secondary antibodies polyclonal rabbit anti-goat immunoglobulins/HRP (Dako, Cat. #P0449). For the development of immunohistochemistry staining, streptavidin- peroxidase (BioConcept, Cat. #71-00-38) and DAB (Sigma-Aldrich, Cat. #D4293-50SET) were used. Primary antibodies were incubated with gentle agitation inside a wet chamber overnight at 4°C. Slides were washed for 20 minutes in PBS-Tween-20 [0,5%] (Sigma-Aldrich, Cat. #P1379) and incubated in darkness for 90 minutes with the secondary antibodies and DAPI (Sigma- Aldrich, Cat. #D9542, diluted 1:2000).
  • Precast gels (Bio-Rad, Cat. #456-1094) were used to separate equalized amounts of protein per sample by SDS-PAGE, under reducing conditions. Proteins were then transferred on nitrocellulose membranes (Biorad, Cat. #170- 4158). Membranes were blocked with 5% w/v nonfat dry milk in PBS for 1 hour at room temperature. Primary antibodies were diluted in the blocking medium and incubated overnight at 4°C. Primary antibodies: CLDN3, (Novus Biologicals, Cat. #NBP1-35668), 1:1000; Anti- ⁇ - Actin ⁇ Peroxidase, (Sigma-Aldrich, Cat. # A3854), 1:50000; Secondary antibodies: anti-rabbit- HRP (Dako, Cat.
  • siRNA candidate screening Candidate siRNAs targeting human claudin-3 mRNA were first identified bioinformatically (human reference sequence used: NM_001306.4). All possible siRNAs were created, and then scored for speficity, cross-reactivity, activity, and off-targets (Axolabs, Kulmbach, Germany). The best candidates were selected and synthetised, containing stabilizing modifications of 2'- fluoro- or 2'O-methyl modifications, and phosphorothioate linkers at the indicated positions (see table 6). The synthetized candidate siRNAs were then transfected into the human PLC liver cell hepatoma cell line (ATCC Cat. No CRL-8024) using lipofectamine-3000.
  • siRNAs were used at a final concentration of 50nM. Two days after transfection, RNA was isolated from the cell cultures, and the levels of remaining human claudin-3 mRNA were quantified using RT-qPCR following the protocols below.34 siRNAs with a knockdown efficacy of 50% or higher were selected for further testing (table 6). Table 7 displays the same identified human claudin-3 siRNA sequences, without their chemically stabilizing modifications.
  • RT-qPCR mRNA expression analysis RNA from snap frozen tissue or cell cultures was extracted using NucleoZOL (Macherey- Nagel, Cat. #740404.200). cDNA was made from either 500 ⁇ g of tissue RNA using the Omniscript reverse transcriptase kit (Qiagen, Cat. #205113).
  • the antibody consists of a heavy chain (SEQ ID NO: 170) and a light chain (SEQ ID NO: 169) as displayed below.
  • SEQ ID NO: 170 a heavy chain
  • SEQ ID NO: 169 a light chain
  • Human hepatocytes and mouse hepatocytes from C57BL/6 and C57BL/6 Cldn3-/- mice were isolated from the liver specimens following a two-step enzymatic perfusion protocol (doi: 10.1172/JCI115207).
  • the viability of the isolated hepatocytes was determined by trypan blue exclusion, and only preparations of over 90% viability were used.
  • the hepatocytes were seeded onto tissue culture plastic coated with rat tail collagen in Dulbecco's modified Eagle medium containing 10% fetal bovine serum, left to attach for 1 to 2 hours, and then washed twice with phosphate-buffered saline (PBS) to remove any remaining non-viable cells from the culture.
  • PBS phosphate-buffered saline
  • the hepatocytes were cultured in arginine-free Williams E medium supplemented with insulin (0.015 IU/mL), hydrocortisone (5 ⁇ mol/L), penicillin (100 IU/mL), streptomycin (100 ⁇ g/mL), glutamine (2 mmol/L), and ornithine (0.4 mmol/L) for 24 hours before use.
  • SNU-449 cells (ATCC#2234) were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 mg/mL streptomycin.
  • Hepa1-6 (ATCC # CRL- 1830) were grown in DMEM supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 mg/mL streptomycin.
  • Flow cytometry for human claudin-3 antibody tests Binding capacity of the Cldn3 antibody was accessed by serial dilution of the stock solution of the anti-Claudin 3 antibody (0.5 mg/ml) in facs buffer exposed to 5.105 human hepatocyte or SNU449 cells for 30 min at 4c. The cells are then rinsed with PBS and exposed to a cyanine5 cojugated secondary anti mouse IgG (Life technologies #A10524) at 1:200 for 30 min at 4c in the darc, before being rinsed with PBS.
  • a cyanine5 cojugated secondary anti mouse IgG (Life technologies #A10524) at 1:200 for 30 min at 4c in the darc, before being rinsed with PBS.
  • a secondary FITC-anti-mouse IgG (ref) was applied to the cells at a dilution of 1:200.
  • the secondary antibody was allowed to bind for 30 minutes at 4°C in the dark.
  • the cells were washed with PBS to eliminate any excess secondary antibody.
  • Cellular data acquisition was performed using a SORP LSRII flow cytometer (BD Pharmingen Inc., San Diego, CA). The acquired data were then analyzed using FlowJo software (Treestar, Inc., Ashland, OR) for flow cytometric analysis.
  • GalNAc-anti Cldn3 Antibody Functionalization The antibody was first buffer exchanged into PBS using a 7K Zeba size-exclusion column, which effectively removed any unwanted buffer components and ensured compatibility with subsequent reactions. To functionalize the antibody, it was reacted with 25 equivalents of DBCO-PEG4-NHS (BroadPharm #BP-22288). The antibody solution in PBS was incubated overnight at room temperature to allow for efficient conjugation between the antibody and the DBCO-PEG4- NHS. Following the conjugation reaction, the functionalized antibody was purified using aPBS equiilibrated 7K Zeba size-exclusion column.
  • This purification step helped to separate the conjugated antibody from any unreacted DBCO-PEG4-NHS and other impurities, resulting in a purified functionalized antibody preparation.
  • the purified functionalized antibody was reacted with 100 equivalents of Trebler GalNAc-Azide (primetech #0079). This reaction facilitated the attachment of Galactose N acetyl (GalNAc) moieties to the functionalized antibody, enabling targeted delivery to GalNAc receptors.
  • the antibody was once again purified, this time using a PBS equilibrated 40K Zeba size-exclusion column.
  • This purification step further separated the conjugated antibody from any unreacted GalNAc-Azide and other remaining impurities, yielding a purified and functionalized antibody ready for downstream applications.
  • Cldn3 KD using GalNAc-anti Cldn3 Antibody Human hepatocytes in 12 well plate were treated with varying concentrations of the GalNAc- anti Cldn3 antibody. Specifically, cells were treated with 0, 1, 10, and 100 nM concentrations of the antibody for a duration of 24 hours. Following the 24-hour treatment period, proteins were extracted from the treated cells using 50 ul of RIPA lysis buffer. The extracted proteins were quantified using the Bio-Rad Protein Assay System (Bio-Rad Laboratory, Melville, NY).
  • RNA sequencing of tissue samples Total RNA was extracted from the liver with NucleoZOL, quantified by a bioanalyzer. The setting of the sequencing run included TruSeq Stranded mRNA, with paired-read ends. Read length was set to 50, and the multiplex level was 1. RNA-seq alignment fastq files were aligned to the mouse reference genome mm10 with hisat2 and transformed into bam files with samTOOLS. The read count matrix was produced from the bam files with the featurecounts function of the R package Rsubread.
  • RNA-seq Differential expression Differentially expressed genes were computed with R package DESeq2. Genes with an adjusted by False Discovery Rate p-value below 0.05 were considered statistically significant for further analysis. Enrichment analysis To determine the pathways to which genes were associated we used Metascape. Statistical significant genes. When more than 2000 were present in a list we took the top 2000 sorted by Fold Change.
  • siRNAs were created from mouse Cldn3 mRNA sequence (NM_009902.4). The bases within the siRNAs were modified for more chemical stability using 2 ⁇ Fluoro- and/or 2 ⁇ O-Methyl residues and phosphorothioate linkages.
  • Claudin-3 targeting siRNA1 (sense strand sequence), Claudin- 3 targeting siRNA2 (sense strand sequence) and AHSA1 control siRNA (sense strand sequence) were conjugated with a triantennary GalNAc cluster (CPG, Primetech). siRNAs were HPLC-purified and lyophilized. For in vivo use, GalNAc siRNAs were diluted with saline solution.
  • the siRNAs were injected subcutaneously at a concentration of 10mg/KG bodyweight. Mice were pre-treated with the siRNAs for two- or four days prior to the experiment. Results Claudin-3 knockout leads to impaired bile acid metabolism, dilution of bile, a higher bile flow rate and impaired uptake of the tracer FITC-Dextran.
  • Wildtype animals expressed the expected BDL phenotype, including dark green coloured bile and visible necrotic patches in liver tissue (Figure 2A). These features were absent in the Cldn3-/- group, indicating a different composition of the bile and significantly ameliorated necrosis.
  • the histological analysis of the liver tissue showed no difference among control groups, which had a normal liver morphology ( Figure 2B).
  • wildtype animals developed tissue necrosis as a typical features of obstructive liver injury (Figure 2B and C), which gained in severity as the cholestasis progressed. Strikingly, mice lacking claudin-3 developed almost no necrosis (Figure 2B and C).
  • ALT alanine aminotransferase
  • AST Aspartate transaminase
  • Alkaline phosphatase levels significantly differed after 12 days post BDL with on average 822,5 U/L in mice and 452,0 U/L in Cldn3-/- mice ( Figure 2F). It was previously described that BDL leads to translocation of bacteria to the liver, being an indicator of tissue injury16.
  • We plated homogenates of livers after BDL and counted the number of colony forming units (CFU). The quantification showed that the number of bacteria that translocated to the liver was significantly lower in Cldn3-/- livers at 2 days post BDL compared to wildtype livers (P 0.01) (Figure 2G). At 7 days post BDL the amount of CFU was similar among groups.
  • TBA levels in gallbladder bile collected 7 days post BDL were 3435 ⁇ mol/L in Cldn3+/+ and only 2611 ⁇ mol/L in Cldn3-/- (P ⁇ 0.01) ( Figure 3B).
  • Liver TBA at 2 days post BDL was on average 308,3 nmol/g in Cldn3+/+ and only 191,8 nmol/g in Cldn3-/- liver (p ⁇ 0.01) ( Figure 3C).
  • Claudin-3 deletion does not majorly alter of the composition of the bile acid pool under normal-or cholestatic conditions During cholestasis, increased hydrophobicity of bile can be important contributor to tissue damage, which depends on the composition of bile with individual bile acid (BA) subtypes.
  • BA bile acid
  • T- ⁇ MCA, T- ⁇ MCA, T- ⁇ MCA and tauro-cholic acid were by far the most abundant bile acid species in the liver bile acid pool ( Figure 4A-C and table 1). Reporting the bile acid amount as nanogram/milligram liver tissue showed that hepatic bile acid levels raise dramatically after 7 days BDL ( Figure 4A). The LC-MS/MS analysis further confirmed our previous observation of reduced liver bile acid levels in Cldn3-/- livers at seven days post BDL ( Figure 3C and 4A). We next calculated the percentages of bile acid subtypes from the whole liver bile acid pool.
  • Cldn3-/- mice have increased periductal oedema but not change in ductular reaction
  • tissue morphology of wildtype and claudin-3 deficient mice post BDL we described the tissue morphology of wildtype and claudin-3 deficient mice post BDL in more detail. Since claudin-3 is highly expressed in cholangiocytes27 and loss of claudin-3 has been previously associated with an impaired of the paracellular barrier for water30,31 , we scored hematoxylin and eosin stained liver sections for oedema in periductal zones.
  • the bile contained in the gallbladder had a normal light-yellow colour (appears lighter / white in the grayscale figure) in Cldn3-/- mice, in contrast to the dark green bile in ANIT challenged Cldn3+/+ ( Figure 8B, appears dark / black in the grayscale figure).
  • Blood ALT ALP and bilirubin levels were significantly lower in Cldn3-/- animals (P ⁇ 0.05) and there was a trend for lower AST levels as well (Figure 8E).
  • Figure 8E Evidentiary for the absence of cholestatic injury were also the significantly lower BA levels.
  • GalNAc siRNA targeting of claudin-3 achieves modest amelioration of cholestatic injury
  • KD knocked down
  • the advantages of this technique are that it produces a stable KD of the target, can be administered subcutaneously, only targets liver hepatocytes and is already in used in clinics33.
  • siRNA1 SEQ ID NO:1, sense strand
  • siRNA2 SEQ ID NO: 2, sense strand
  • siRNAs were scored for target specificity, intra- and inter- species cross-reactivity and activity. The best siRNA candidates were then synthetised (Axolabs, Kulmbach, Germany). siRNA sequences contained chemically stabilising additions of 2'-fluoro- or 2'O-methyl modifications, or phosphorothioate linkers at the positions indicated in table 6. Next, the synthetised siRNA candidates were evaluated for their efficiency to knockdown human claudin-3, in cultures of the human liver cell line PLC (ATCC Cat. No CRL-8024).
  • siRNAs that knockdown 50% or more of human claudin-3 mRNA displayed without modifications. The experiment was done twice, resulting in comparable results.
  • siRNA sequences that knocked down human claudin-3 with an efficacy of equal- or greater than 50% The best performing siRNAs will be now used further for confirming- and de-risking studies, following a typical drug development process.
  • Figure 10 presents the binding analysis of the anti claudin-3 antibody UB-VS003 against Human SNU449 cells and hepatocytes, as well as mouse Hepa1-6 cells. The results demonstrate a consistent trend in antibody binding for all cell types. For Human SNU449 cells and hepatocytes, the control samples showed no detectable binding (0%). However, as the antibody concentration increased, a progressive increase in binding was observed. At a dilution of 1/400, the binding percentage was approximately 3%. This increased to 7% at a dilution of 1/200, 12% at 1/100, and reached a maximum of 25% at 1/50 dilution.
  • the ratio of claudin-3 to beta-actin was quantified to assess the relative abundance of claudin-3 protein.
  • the ratio of claudin-3 to beta-actin was set at 100%, indicating that claudin-3 was fully expressed under normal conditions.
  • a dose-dependent decrease in the claudin-3/beta-actin ratio was observed, reflecting a reduction in claudin-3 protein levels.
  • the claudin-3/beta-actin ratio decreased to approximately 45% compared to the control sample. This reduction became more pronounced as the antibody concentration increased further.
  • the Transcription Factor MEF2 Is a Novel Regulator of Gsta Gene Class in Mouse MA-10 Leydig Cells. Endocrinology 2015;156:4695–4706.
  • FXR Isoforms Control Different Metabolic Functions in Liver Cells via Binding to Specific DNA Motifs. Gastroenterology 2020;159:1853-1865.e10.
  • Baier FA Sánchez-Taltavull D, Yarahmadov T, Castellà CG, Jebbawi F, Keogh A, Tombolini R, Odriozola A, Dias MC, Deutsch U, Furuse M, Engelhardt B, Zuber B, Odermatt A, Candinas D, Stroka D. Loss of Claudin-3 Impairs Hepatic Metabolism, Biliary Barrier Function, and Cell Proliferation in the Murine Liver. Cell Mol Gastroenterol Hepatol 2021;S2352345X21000746. 28. Croce AC, Ferrigno A, Santin G, Piccolini VM, Bottiroli G, Vairetti M.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Public Health (AREA)
  • Biotechnology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Zoology (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Epidemiology (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne une méthode pour prévenir, retarder la progression ou traiter la cholestase et/ou la fibrose associée à la cholestase.
PCT/EP2023/067574 2022-06-30 2023-06-28 Méthodes pour prévenir, retarder la progression ou traiter la cholestase et/ou la fibrose associée à la cholestase WO2024003102A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP22182112.7 2022-06-30
EP22182112 2022-06-30

Publications (1)

Publication Number Publication Date
WO2024003102A1 true WO2024003102A1 (fr) 2024-01-04

Family

ID=82492383

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2023/067574 WO2024003102A1 (fr) 2022-06-30 2023-06-28 Méthodes pour prévenir, retarder la progression ou traiter la cholestase et/ou la fibrose associée à la cholestase

Country Status (1)

Country Link
WO (1) WO2024003102A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006044335A2 (fr) * 2004-10-14 2006-04-27 The Board Of Trustees Of The University Of Arkansas Traitement du cancer ovarien et utérin par l'entérotoxine clostridium perfringens
EP2103628A1 (fr) * 2006-12-14 2009-09-23 Forerunner Pharma Research Co., Ltd. Anticorps monoclonal anti-claudine 3, et traitement et diagnostic du cancer au moyen d'un tel anticorps

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006044335A2 (fr) * 2004-10-14 2006-04-27 The Board Of Trustees Of The University Of Arkansas Traitement du cancer ovarien et utérin par l'entérotoxine clostridium perfringens
EP2103628A1 (fr) * 2006-12-14 2009-09-23 Forerunner Pharma Research Co., Ltd. Anticorps monoclonal anti-claudine 3, et traitement et diagnostic du cancer au moyen d'un tel anticorps

Non-Patent Citations (48)

* Cited by examiner, † Cited by third party
Title
"Pharmaceutical Dosage Forms and Drug Delivery Systems", 1999, LIPPINCOTT WILLIAMS & WILKINS
"Pharmaceutical Dosage Forms", 1980, MARCEL DECKER
"Remington: The Science and Practice of Pharmacy", 1995, MACK PUBLISHING COMPANY
BAIER FASANCHEZ-TALTAVULL DYARAHMADOV TCASTELLA CGJEBBAWI FKEOGH ATOMBOLINI RODRIOZOLA ADIAS MCDEUTSCH U: "Loss of Claudin-3 Impairs Hepatic Metabolism, Biliary Barrier Function, and Cell Proliferation in the Murine Liver", CELL MOL GASTROENTEROL HEPATOL, 2021
BAIER FELIX ALEXANDER ET AL: "Loss of Claudin-3 Impairs Hepatic Metabolism, Biliary Barrier Function, and Cell Proliferation in the Murine Liver", CMGH CELLULAR AND MOLECULAR GASTROENTEROLOGY AND HEPATOLOGY, vol. 12, no. 2, 1 January 2021 (2021-01-01), pages 745 - 767, XP093008515, ISSN: 2352-345X, DOI: 10.1016/j.jcmgh.2021.04.003 *
CAI S-YBOYER JL: "The role of bile acids in cholestatic liver injury", ANN TRANSL MED, vol. 9, 2021, pages 737 - 737
CHANG M-L: "Comparison of murine cirrhosis models induced by hepatotoxin administration and common bile duct ligation", WORLD J GASTROENTEROL, vol. 11, 2005, pages 4167
CHIHARA MIKEBUCHI ROTSUKA SICHII OHASHIMOTO YSUZUKI ASAGA YKON Y: "Mice Stage-Specific Claudin 3 Expression Regulates Progression of Meiosis in Early Stage Spermatocytes 1", BIOL REPROD, vol. 89, 1 July 2013 (2013-07-01)
COMEGLIO PFILIPPI SSARCHIELLI EMORELLI ACELLAI ICORCETTO FCOMO CMANESCHI EPINI AADORINI L: "Anti-fibrotic effects of chronic treatment with the selective FXR agonist obeticholic acid in the bleomycin-induced rat model of pulmonary fibrosis", J STEROID BIOCHEM MOL BIOL, vol. 168, 2017, XP029936739, DOI: 10.1016/j.jsbmb.2017.01.010
CROCE ACFERRIGNO ASANTIN GPICCOLINI VMBOTTIROLI GVAIRETTI M: "Autofluorescence of liver tissue and bile: Organ functionality monitoring during ischemia and reoxygenation: AUTOFLUORESCENCE OF LIVER TISSUE AND BILE", LASERS SURG MED, vol. 46, 2014, pages 412 - 421
DI-LUOFFO MBROUSSEAU CBERGERON FTREMBLAY JJ: "The Transcription Factor MEF2 Is a Novel Regulator of Gsta Gene Class in Mouse MA-10 Leydig Cells", ENDOCRINOLOGY, vol. 156, 2015, pages 4695 - 4706
FICKERT PPOLLHEIMER MJOSTERREICHER CHTRAUNER M: "Animal Models for the Study of Human Disease", ELSEVIER, article "Animal Models of Cholestasis", pages: 331 - 349
GEORGIEV PNAVARINI AAELORANTA JJLANG KSKULLAK-UBLICK GANOCITO ADAHM FJOCHUM WGRAF RCLAVIEN P-A: "Cholestasis protects the liver from ischaemic injury and post-ischaemic inflammation in the mouse", GUT, vol. 56, 2007, pages 121 - 128
GOMEZ CSTUCHELI SKRATSCHMAR DVBOUITBIR JODERMATT A: "Development and Validation of a Highly Sensitive LC-MS/MS Method for the Analysis of Bile Acids in Serum, Plasma, and Liver Tissue Samples", METABOLITES, vol. 10, 9 July 2020 (2020-07-09)
GROSSE BRIGITTE ET AL: "Build them up and break them down : Tight junctions of cell lines expressing typical hepatocyte polarity with a varied repertoire of claudins", TISSUE BARRIERS, vol. 1, no. 4, 4 June 2013 (2013-06-04), pages e25210, XP093008977, DOI: 10.4161/tisb.25210 *
HIRSCHFIELD GMGERSHWIN MESTRAUSS RMAYO MJLEVY CZOU BJOHANNS JNNANE IPDASGUPTA BLI K: "PURIFI Study Group. Ustekinumab for patients with primary biliary cholangitis who have an inadequate response to ursodeoxycholic acid: A proof-of-concept study", HEPATOL BALTIM MD, vol. 64, 2016, pages 189 - 199
HOOVER, JOHN E.: "Remington's Pharmaceutical Sciences", 1975, MACK PUBLISHING CO.
HUANG YU-HUNG ET AL: "Claudin-3 gene silencing with siRNA suppresses ovarian tumor growth and metastasis", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, vol. 106, no. 9, 3 March 2009 (2009-03-03), pages 3426 - 3430, XP002547909, ISSN: 0027-8424, [retrieved on 20090210], DOI: 10.1073/PNAS.0813348106 *
HUANG YU-HUNG ET AL: "Claudin-3 gene silencing with siRNA suppresses ovarian tumor growth and metastasis-Supplemental materials", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 106, no. 9, 3 March 2009 (2009-03-03), pages 3426 - 3430, XP093008447, ISSN: 0027-8424, DOI: 10.1073/pnas.0813348106 *
JAVITT NORMAN B.: "Hepatic bile formation: bile acid transport and water flow into the canalicular conduit", AMERICAN JOURNAL OF PHYSIOLOGY - GASTROINTESTINAL AND LIVER PHYSIOLOGY, vol. 319, no. 5, 1 November 2020 (2020-11-01), US, pages G609 - G618, XP093008452, ISSN: 0193-1857, DOI: 10.1152/ajpgi.00078.2020 *
JONES DBOUDES PFSWAIN MGBOWLUS CLGALAMBOS MRBACON BRDOERFFEL YGITLIN NGORDON SCODIN JA: "Seladelpar (MBX-8025), a selective PPAR-δ agonist, in patients with primary biliary cholangitis with an inadequate response to ursodeoxycholic acid: a double-blind, randomised, placebo-controlled, phase 2, proof-of-concept study", LANCET GASTROENTEROL HEPATOL, vol. 2, 2017, pages 716 - 726, XP009530038, DOI: 10.1016/S2468-1253(17)30246-7
JOSHI NRAY JLKOPEC AKLUYENDYK JP: "Dose-dependent effects of alpha-naphthylisothiocyanate disconnect biliary fibrosis from hepatocellular necrosis", J BIOCHEM MOL TOXICOL, vol. 31, 2017, pages 1 - 7
LEVY CPETER JANELSON DRKEACH JPETZ JCABRERA RCLARK VFIRPI RJMORELLI GSOLDEVILA-PICO C: "Pilot study: fenofibrate for patients with primary biliary cirrhosis and an incomplete response to ursodeoxycholic acid: Pilot study: fenofibrate with UDCA for primary biliary cirrhosis", ALIMENT PHARMACOL THER, vol. 33, 2011, pages 235 - 242, XP071542085, DOI: 10.1111/j.1365-2036.2010.04512.x
LIU JHE Y-YCHIGNELL CFCLARK JMYERS PSAAVEDRA JEWAALKES MP: "Limited protective role of V-PYRRO/NO against cholestasis produced by alpha-naphthylisothiocyanate in mice", BIOCHEM PHARMACOL, vol. 70, 2005, pages 144 - 151
MERLEN GKAHALE NURSIC-BEDOYA JBIDAULT-JOURDAINNE VSIMERABET HDOIGNON ITANFIN ZGARCIN IPEAN NGAUTHEROT J: "TGR5-dependent hepatoprotection through the regulation of biliary epithelium barrier function", GUT, vol. 69, 2020, pages 146 - 157
MERRELL MDNYAGODE BACLARKE JDCHERRINGTON NJMORGAN ET: "Selective and Cytokine-Dependent Regulation of Hepatic Transporters and Bile Acid Homeostasis during Infectious Colitis in Mice", DRUG METAB DISPOS, vol. 42, 2014, pages 596 - 602
MICHAEL D THOMPSON ET AL: "[beta]-Catenin regulation of farnesoid X receptor signaling and bile acid metabolism during murine cholestasis", HEPATOLOGY, JOHN WILEY & SONS, INC, US, vol. 67, no. 3, 26 January 2018 (2018-01-26), pages 955 - 971, XP071563066, ISSN: 0270-9139, DOI: 10.1002/HEP.29371 *
MOGHADAMRAD SMCCOY KDGEUKING MBSAGESSER HKIRUNDI JMACPHERSON AJDE GOTTARDI A: "Attenuated portal hypertension in germ-free mice: Function of bacterial flora on the development of mesenteric lymphatic and blood vessels: HEPATOLOGY", HEPATOLOGY, vol. 61, 2015, pages 1685 - 1695
MYERS RPSWAIN MGLEE SSSHAHEEN AAMBURAK KW: "B-cell depletion with rituximab in patients with primary biliary cirrhosis refractory to ursodeoxycholic acid", AM J GASTROENTEROL, vol. 108, 2013, pages 933 - 941
NEEDLEMANWUNSCH, J. MOL. BIOL., vol. 48, 1970, pages 443
NEVENS FANDREONE PMAZZELLA GSTRASSER SIBOWLUS CINVERNIZZI PDRENTH JPHPOCKROS PJREGULA JBEUERS U: "A Placebo-Controlled Trial of Obeticholic Acid in Primary Biliary Cholangitis", N ENGL J MED, vol. 375, 2016, pages 631 - 643, XP055723834, DOI: 10.1056/NEJMoa1509840
PEARSONLIPMAN, PROC. NAT. ACAD. SCI., vol. 85, 1988, pages 2444
PENNO CAARSENIJEVIC DDA CUNHA TKULLAK-UBLICK GAMONTANI J-PODERMATT A: "Quantification of multiple bile acids in uninephrectomized rats using ultra-performance liquid chromatography-tandem mass spectrometry", ANAL METHODS, vol. 5, 2013, pages 1155
PRADHAN-SUNDD TIRTHADIPA ET AL: "Dysregulated Bile Transporters and Impaired Tight Junctions During Chronic Liver Injury in Mice", GASTROENTEROLOGY, ELSEVIER INC, US, vol. 155, no. 4, 30 June 2018 (2018-06-30), pages 1218, XP085495439, ISSN: 0016-5085, DOI: 10.1053/J.GASTRO.2018.06.048 *
PRADHAN-SUNDD TIRTHADIPA ET AL: "Supplemental Material to: Dysregulated bile transporters and impaired tight junctions promote chronic liver injury in mice", 30 June 2018 (2018-06-30), XP093008627, Retrieved from the Internet <URL:https://europepmc.org/article/pmc/pmc6174089> [retrieved on 20221215] *
RAHIMPOUR SNASIRI-TOOSI MKHALILI HEBRAHIMI-DARYANI NNOURI-TAROMLOU MKAZIZI Z: "A Triple Blinded, Randomized, Placebo-Controlled Clinical Trial to Evaluate the Efficacy and Safety of Oral Vancomycin in Primary Sclerosing Cholangitis: a Pilot Study", J GASTROINTEST LIVER DIS JGLD, vol. 25, 2016, pages 457 - 464
RAINTEAU DLEFEVRE GDE CHAISEMARTIN LCHOLLET-MARTIN SGAOUAR FADMANE F-HSIMON TPOUPON R: "A Placebo-Controlled Trial of Bezafibrate in Primary Biliary Cholangitis", N ENGL J MED, vol. 378, 2018, pages 2171 - 2181
RAMOS PITTOL JMMILONA AMORRIS IWILLEMSEN ECLVAN DER VEEN SWKALKHOVEN EVAN MIL SWC: "FXR Isoforms Control Different Metabolic Functions in Liver Cells via Binding to Specific DNA Motifs", GASTROENTEROLOGY, vol. 159, 2020, pages 1853 - 1865
SANTIAGO PSCHEINBERG ARLEVY C: "Cholestatic liver diseases: new targets, new therapies.", THER ADV GASTROENTEROL, vol. 11, 2018
SMITHWATERMAN, ADV. APPL. MATH., vol. 2, 1981, pages 482
SPRINGER ADDOWDY SF: "GalNAc-siRNA Conjugates: Leading the Way for Delivery of RNAi Therapeutics", NUCLEIC ACID THER, vol. 28, 2018, pages 109 - 118, XP055555952, DOI: 10.1089/nat.2018.0736
TABIBIAN JHGOSSARD AEL-YOUSSEF MEATON JEPETZ JJORGENSEN RENDERS FBTABIBIAN ALINDOR KD: "Prospective Clinical Trial of Rifaximin Therapy for Patients With Primary Sclerosing Cholangitis", AM J THER, vol. 24, 2017, pages e56 - e63
TANAKA HIMASATO MYAMAZAKI YMATSUMOTO KKUNIMOTO KDELPIERRE JMEYER KZERIAL MKITAMURA NWATANABE M: "Claudin-3 regulates bile canalicular paracellular barrier and cholesterol gallstone core formation in mice", J HEPATOL, vol. 69, 2018, pages 1308 - 1316, XP085535551, DOI: 10.1016/j.jhep.2018.08.025
TANAKA HIROO ET AL: "Claudin-3 regulates bile canalicular paracellular barrier and cholesterol gallstone core formation in mice", JOURNAL OF HEPATOLOGY, ELSEVIER, AMSTERDAM, NL, vol. 69, no. 6, 11 September 2018 (2018-09-11), pages 1308 - 1316, XP085535551, ISSN: 0168-8278, DOI: 10.1016/J.JHEP.2018.08.025 *
TSUDA MMORITOKI YLIAN Z-XZHANG WYOSHIDA KWAKABAYASHI KYANG G-XNAKATANI TIERLING JLINDOR K: "Biochemical and immunologic effects of rituximab in patients with primary biliary cirrhosis and an incomplete response to ursodeoxycholic acid", HEPATOL BALTIM MD, vol. 55, 2012, pages 512 - 521
YAMAGA KMUROTA HTAMURA AMIYATA HOHMI MKIKUTA JISHII MTSUKITA SKATAYAMA I: "Claudin-3 Loss Causes Leakage of Sweat from the Sweat Gland to Contribute to the Pathogenesis of Atopic Dermatitis", J INVEST DERMATOL, vol. 138, 2018, pages 1279 - 1287
ZHANG TYU FGUO LCHEN MYUAN XWU B: "Small Heterodimer Partner Regulates Circadian Cytochromes p450 and Drug-Induced Hepatotoxicity", THERANOSTICS, vol. 8, 2018, pages 5246 - 5258
ZHOU YAXIAN ET AL: "Development of Triantennary N-Acetylgalactosamine Conjugates as Degraders for Extracellular Proteins", 19 August 2020 (2020-08-19), pages 1 - 28, XP055797480, Retrieved from the Internet <URL:https://s3-eu-west-1.amazonaws.com/itempdf74155353254prod/12824834/Development_of_Triantennary_N-Acetylgalactosamine_Conjugates_as_Degraders_for_Extracellular_Proteins_v1.pdf> [retrieved on 20210421], DOI: 10.26434/chemrxiv.12824834.v1 *

Similar Documents

Publication Publication Date Title
Rosenne et al. In vivo suppression of NK cell cytotoxicity by stress and surgery: glucocorticoids have a minor role compared to catecholamines and prostaglandins
Li et al. C3a receptor antagonist ameliorates inflammatory and fibrotic signals in type 2 diabetic nephropathy by suppressing the activation of TGF-β/smad3 and IKBα pathway
JP2020523992A (ja) Ｇタンパク質共役受容体媒介症状を治療するための組成物および方法
WO2020257648A1 (fr) Ciblage microluminal de cellules cancéreuses
US7867496B2 (en) Use of TGF-β antagonists to limit nephrotoxicity of immunosuppressive agents
JP5819285B2 (ja) 子癇前症の検出を補助する方法またはそのリスクの推定を補助する方法
KR20140063825A (ko) 클로스트리듐 디피실의 주요 외독소 tcda 및 tcdb에 대한 중화 항체
US20200009248A1 (en) Targeting tgr5 to treat disease
WO2020169472A2 (fr) Procédés d&#39;induction de changements phénotypiques dans des macrophages
JP2019532049A (ja) Ｄｒｐ１阻害を通じたｐｃｓｋ９およびｌｄｌｒのモジュレーション法
Barratt et al. Treatment of IgA nephropathy: evolution over half a century
Claus et al. The TWEAK/Fn14 pathway is required for calcineurin inhibitor toxicity of the kidneys
KR20180135928A (ko) 간 질환을 치료하거나 예방하는 방법
Chen et al. IRF-4 deficiency reduces inflammation and kidney fibrosis after folic acid-induced acute kidney injury
WO2012064967A2 (fr) Le récepteur activateur du ligand nf-kb, issu de cellules cancéreuses, entraîne des métastases des os et des tissus mous
JP6941565B2 (ja) がん転移を処置または防止するのに有用な化合物および組成物、ならびにこれらの使用法
Shen et al. AUY922 induces retinal toxicity through attenuating TRPM1
US11564906B2 (en) Methods and compositions for the treatment of retinopathy and other ocular diseases
WO2024003102A1 (fr) Méthodes pour prévenir, retarder la progression ou traiter la cholestase et/ou la fibrose associée à la cholestase
JP7173589B2 (ja) 肝臓病の処置における使用のための抗ｃｃｌ２４（エオタキシン２）抗体
CN115068612A (zh) Drd2抑制剂在制备治疗与肝脏纤维化有关的疾病的药物中的应用
US20230136299A1 (en) Treatment of liver failure
US20210017268A1 (en) Methods for treating alcoholic liver disease, alcohol-induced brain injury and reducing alcohol addiction
Ceci et al. Prolonged administration of melatonin ameliorates liver phenotypes in cholestatic murine model
WO2020077120A1 (fr) Blocage de rgmb pour traiter une maladie intestinale inflammatoire et une colite

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23736274

Country of ref document: EP

Kind code of ref document: A1